Inaccurate repair of DSBs can lead to such potentially lethal events as genomic rearrangements or deletion. Unrepaired DSBs can lead directly to chromosome loss and indirectly may be mediators of untargeted DNA metabolic events including enhanced recombination or replication arrest. We have utilized the site specific endonuclease HO and the MAT switching locus (YZ junction) from the yeast S. cerevisiae to investigate the molecular consequences of extrachromasomally induced DSBs. A 45 bp YZ junction fragment flanked by nonhomologous bacterial DNA sequences was cloned into a selectable high copy 2u vector and transformed into a nonswitching deploid yeast strain ACY522. This strain was subsequently transformed with a second selectable plasmid (pGALHOT) containing the HO endonuclease under GAL control. In vivo breakage of the YZ junction DSB cut site was monitored at various times following galactose addition using Southern blot analysis. DS breakage at the YZ junction was initially observed at 4 hours and was maximal at 10 hours following galactose addition. YZ cutting of the 2u target plasmid was incomplete with >50% supercoiled plasmid remaining at 10 hours after galactose addition. Since >80% of the cells maintain the HO plasmid pGALHOT during the galactose time course, a significant fraction of the cells with DSBs also contain unbroken plasmid. Plating of cells containing the HO and YZ target plasmids under selection to minimal galactose plates results in a severe inhibition of growth producing microcolonies of elongated filamentous cells. Glucose rescue of these microcolonies suggest that the majority of these cells are dead. Morphological analysis of unsynchronized galactose grown 2uYZ and PGALHOT containing cells show an increased number of doublet cells characteristic of a radiation induced cell cycle blockage in G2. Cytological staining of cell nuclei of doublet cells using DAPI or giemsa stains indicate a dumbbell morphology with the nucleus trapped in the bud isthmus. These results are consistent with DSBs mediating cellular arrest in trans.